Field of the Invention
This invention relates to single-reduced tunicamycin related compounds and double-reduced tunicamycin related compounds, and methods of making these tunicamycin related compounds. This invention also relates to antibacterial compositions containing a combination of one or more prior art antibiotics and either one or more of the single-reduced tunicamycin related compounds or one or more of the double-reduced tunicamycin related compounds. This invention also relates to the use of these tunicamycin related compounds alone or in combination with other antibiotics to kill bacterial pathogens in animals.
Description Of Related Art
Antimicrobial resistance is considered one of the most serious health threats to both animals and humans. The penicillins were developed by Howard Florey and coworkers in collaboration with the U.S.D.A. in the 1940's, and, despite the rise of resistance, they are still used to treat a wide range of bacteria. Currently, 60% of antibiotics are used for agricultural purposes. The introduction of second generation beta-lactams and of lactam-clavulinic acid combinations initially overcame resistance to some extent, but the rise in pathogens with beta-lactam resistance is now a major concern.
Bacteria often develop resistance to β-lactam antibiotics by expressing a β-lactamase enzyme that attacks the β-lactam ring, thereby rending the antibiotic inactive. Noticeably, Staphylococcus aureus lacks a β-lactamase resistance enzyme and thus are unable to degrade β-lactam antibiotics. However, the methicillin-resistant S. aureus (MRSA) acquired an alternative protein, penicillin-binding protein (Pbp2a) that inhibits β-lactam antibiotics. Recent studies have demonstrated that MRSA can be rendered susceptible to β-lactam antibiotics again, if the bacterium's teichoic acid biosynthesis is blocked. Several cell wall teichoic acid (WTA) inhibitors exist, such as Targocil, L275, L638, L524, and L555, although most of these compounds have efficacy and toxicity issues. Recently, 2.8 million small molecules were screened for effective WTA inhibitors, and it was determined that two synthetic chemicals (tarocin A (FIG. 1A) and tarocin B (FIG. 1B)) block the first step of teichoic acid synthesis by inhibiting the TarO protein. See, Lee, et al., Science Translat. Med. 8:329-329 (ra32 9) (2016). A derivative, tarocin A1, displays complete depletion of the cell wall teichoic acid polymer.
In addition to tarocin A1, tunicamycins display complete depletion of the cell wall teichoic acid polymer. Tunicamycins have known biological activity against eukaryotes by blocking the first step of protein N-glycosylation, ultimately leading to cell death. Tunicamycins inhibit UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT), a membrane-bound protein that catalyzes the biosynthesis of N-acetylglucosamine-linked dolichol pyrophosphate. This “lipid-linked sugar” is then further modified by subsequent glycosylations, before transfer of the mature N-glycan chain to the Asn-X-Ser/Thr consensus sequence on nascent N-glycoproteins.
Tunicamycins restore β-lactam efficacy against MRSA by inhibiting the formation of the bacterial cell WTA. See, Campbell, et al., ACS Chem. Biol. 6:106-116 (2011). Tunicamycins do this by inhibiting TagO, the first enzyme in WTA biosynthesis. Tunicamycins are the more potent than tarocins and improve the antibacterial activity of oxacillin by 64-fold (Lee, et al., (2016)). Lee, et al., (2016) demonstrated that tunicamycins have potent whole-cell WTA pathway-specific inhibitory effects at ≤0.1 mM, whereas tarocin A and tarocin B are notably less active (3 to 26 mM). Furthermore, the tarocins identified by Lee, et al. (2016)) have much less cytotoxicity against human HeLa cells (inhibitory concentration (IC50), >100 mM) compared to tunicamycins (IC50, 0.2 mM). Tunicamycins are extremely toxic to eukaryotic cells and cannot be used in humans and other animals
Tunicamycins are an analog of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc). See Table 1, infra. As such, they are promiscuous inhibitors of many bacterial glucosyltransferases, such as wall teichoic acid biosynthesis (TagO) and peptidoglycan biosynthesis (MraY) (Mizuno, et al., J. Antibiot. 24:896-899 (1971)). Tunicamycins also inhibit enzymes involved in N-linked protein glycosylation in yeast (Alg7) (Kukuruzinska, et al., Ann. Rev. Biochem. 56:915-944 (1987)) and in human (DPAGT1) (Lehle et al., Angew. Chem. Int. Ed. Engl. 45:6802-18 (2006); Bretthauer, Curr. Drug Targets 10:477-82 (2009)). As such, Lee, et al. (2016) states that tarocins and other inhibitors of early wall teichoic acid biosynthesis “ . . . may provide an important new strategy to develop Gram-positive bactericidal β-lactam combination agents that are active against methicillin-resistant staphylococci” (Id., p9). Lee, et al. (2016) further states “ . . . tunicamycin's cytotoxicity precluded it as a viable chemical starting point to consider as a β-lactam potentiation agent, tarocins provided an attractive alternative therapeutic candidate because they lacked cytotoxicity and intrinsic bioactivity as a single agent.” (Id., p3)
Tunicamycins are a family of nucleotide sugar analogs produced by several Streptomyces species (Takatsuki, et al., J. Antibiot. (Tokyo) 24:215-23 (1971)). The biosynthesis of tunicamycin has been studied previously (Farha, et al., ACS Chem. Biol. 8, 226-233 (2012); Endl, et al. (1983)), as has the genomic requirements (Vinogradov, et al. (2006); Weidenmaier and Peschel, Nat. Rev. Microbiol. 6:276-287 (2008)), and the mechanism of action (Holland, et al. (2011); Tsvetanova and Price, Anal. Biochem. 289:147-156 (2001); Tamura, G., Japan Scientific Press, Tokyo (1982); Kimura and Bugg, Nat. Prod. Reports 20:252-273 (2003)). Twelve tunicamycin biosynthetic genes (tunA to tunL) have been identified in S. chartreusis NRRL B-3882 and S. clavuligerus NRRL 3585 and are also present on the sequenced genome of Actinosynnema mirum DSM 44827, although with a truncated tunL gene (Chen, et al Protein Cell. 1:1093-105 (2010); Wyszynski, et al, Chem. Sci. 1: 581; (2010)). Non-modified, natural tunicamycins are produced commercially via fermentation, usually by the commercial strain S. chartreusis NRRL B-3882 or S. lysosuperificus ATCC 31396. Tunicamycin structures are highly unusual but well characterized (Lee, et al. (2016); Campbell, et al. (2011); Li and Yu, Angewandte Chem. 54:6618-6621 (2015); Navarre and Schneewind (1999)); and are composed of uracil, N-acetylglucosamine (GlcNAc), an amide-linked fatty acid, and a unique 11-carbon 2-aminodialdose sugar called tunicamine Naturally-occurring tunicamycin exists as a mixture of ten or more individual components with different N-linked acyl chains (Campbell, et al. (2011)), and several structurally related compounds also exist. Mycospocidins (from S. bobiliae) (Neuhaus and Baddiley, Microbiol. Mol. Biol. Rev. 67:686-723 (2003)), streptovirudins (S. griseoflavus subsp. thuringiensis) (Brown, et al., Chem. Biol. 15:12-21 (2008); Soldo, et al., Microbiology 148:2079-2087 (2002)), antibiotics MM 19290, (S. clavuligerus NRRL B-3585) and 24010 (from an unidentified streptomycete) (Swoboda, et al., Chem Bio Chem 11:35-45 (2010); Campbell, et al., ACS Chem. Biol. 6, 106-116 (2011)), and corynetoxins (Clavibacter toxicus, also called Corynebacterium rathayi) (Brown, et al., Proc. Natl. Acad. Sci. U.S.A. 109:18909-18914 (2012)) are structurally akin to the tunicamycins, differing only in the N-acyl moiety and/or substitution of 5,6-dihydrouracil for the uracil group. More recently, a new group of related compounds was identified, and called quinovosamycins, from S. niger NRRL B-3857 that are identical to the tunicamycins except that the α-1″-GlcNAc headgroup is replaced by α-1″-QuiNAc (Price, et al., J. Antibiotics 69:637-46 (2016)). Price, et al. (2016) also identified new bacterial strains that contain the Tun biosynthetic operon that confers the ability to produce tunicamycins. Using the tunB and tunD biosynthetic gene sequences as probes of an actinomycetes genomic library, seven microorganisms with the potential for tunicamycin biosynthesis were identified, four of which are previously unreported. These strains are Streptomyces sp. NRRL F-4474, S. niger NRRL B-3857 (formally Chainia nigra), Streptomyces sp. PCS3-D2, and Nocardia nova SH22a, a bacterium capable of degrading gutta-percha. Other strains reported to produce tunicamycins are S. lysosuperificus ATCC 31396 (Takatsuki, et al. (1971)), S. chartreusis NRRL B-12338 and S. chartreusis NRRL B-3882 (Doroghazi, et al., J. Bacteriol. 193:7021-2 (2011)), Clavibacter michiganensis ssp. Michiganensis (Holtmark, et al., J. Appl. Microbiol. 102:416-23 (2007)), and S. torulosus T-4 (Atta, H. M., J. Saudi Chem. Soc. 19:12-22 (2015)).
Because bacteria are becoming resistant to antibiotics currently on the market, a need exists for new antibiotics that are effective. Because naturally-produced tunicamycin is toxic to prokaryotes and eukaryotes, it cannot be used as an antibiotic. However, modifying tunicamycin to be non-toxic against eukaryotic cells but retain toxicity against prokaryotic cells will result in compounds that are extremely valuable antibiotic for use in animals, including humans.